GB 29704-2013Determination of Cyromazine and Melamine residues in animal derived food by Ultra Performance Liquid Chromatography-tandem Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

6.1.1 Eggs

Fresh or frozen egg white or test, peeled, and homogenized.

--- the test sample taken after homogenization, as the feed try.

--- blank sample taken after homogenization, as a blank sample.

--- blank sample taken after homogenization, adding a suitable concentration of the standard working solution, is added as a blank sample.

6.1.2 Chicken muscle meat and kidney

Fresh or frozen blank or test tissue, minced, and homogenized.

--- the test sample taken after homogenization, as the feed try.

--- blank sample taken after homogenization, as a blank sample.

--- blank sample taken after homogenization, adding a suitable concentration of the standard working solution, is added as a blank sample.

Save 6.2 sample

Or less at -20 °C.

Determination Step 7

7.1 Preparation of matrix-matched standard curve

The precise amount of 10µg/mL cyromazine and melamine working standard solutions amount, diluted with 95% acetonitrile in water to prepare a concentrated it is a series of mixed standard solution 2,5,10,20,50 and 100ng/mL and 1.0 mL from each, are extracted by dissolution, purification and drying 6 parts of the blank sample residue was filtered, for performance liquid chromatography - tandem mass spectrometry. A characteristic ion mass chromatogram peak area for the vertical Coordinate matrix-matched control solution concentration as the abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.2 Extract

Sample Weigh 2g ± 0.02g, in 50mL centrifuge tube, 3% trichloroacetic acid solution was added 20mL, vortexed 30s, ultrasonic 10min, allowed to stand for 10min, 10000r/min centrifugal 10min, the supernatant was set aside.

7.3 Purification

MCX column washed with methanol and 3mL 3mL water activation, 10mL take stock solution through the column, 3mL plus 2% formic acid, methanol 3 mL rinsed, drained, with 5% ammoniated methanol 3mL elution, drained, eluate dry nitrogen 50 °C, with 95% aqueous acetonitrile the residue was dissolved 1.0mL, membrane filtration, ultra for liquid chromatography - tandem mass spectrometry.

7.4 Determination

7.4.1 Chromatographic conditions

7.4.1.1 Column. BEHHILIC column (50mm × 2.1mm, particle size 1.7µm), or equivalent person.

7.4.1.2 Mobile phase. A. acetonitrile; B. 0.1mol/L ammonium acetate solution.

7.4.1.3 Gradient. 0min ~ 2.5min, 95% A linear gradient to 80% A; 2.5min ~ 4min, maintaining 95% A.

7.4.1.4 flow rate. 0.3mL/min.

7.4.1.5 Column temperature. 30 °C.

7.4.1.6 Injection volume. 10µL.

7.4.2 MS conditions

7.4.2.1 ion source. electrospray ionization source.

7.4.2.2 Scan mode. positive ion scan.

7.4.2.3 Detection mode. multiple reaction monitoring.

7.4.2.4 ionization voltage. 3.0kV.

7.4.2.5 source temperature. 110 °C.

7.4.2.6 atomizing temperature. 350 °C.

7.4.2.7 Cone gas flow rate. 50L/h.

7.4.2.8 atomizing gas flow rate. 650L/h.

7.4.2.9 qualitative, cone voltage and collision energy of ions and the corresponding quantified. see Table 1.

Table 1 Qualitative and quantitative ion pair and the corresponding cone voltage and collision energy

drug

Qualitative ion pair m/z Quantitative ion pair m/z Cone voltage Collision energy eV Cyromazine 166.9 > 84.6 166.9 > 124.8 166.9 > 84.6 35 Melamine 126.9 > 84.7 126.9 > 67.7 126.9 > 84.7 30 twenty two

7.4.3 Assay

Take a sample solution and a matrix matching the corresponding standard solution, as a single or multi-point calibration, external standard method. Standard solution and the sample solution Peak area cyromazine and melamine should be within the linear range of the detection instrument. Ion in the sample solution with the matrix matching the relative abundance of Compared with a standard relative abundance of ions in solution, in accordance with Table 2. Chromatographic quality standard solution and sample solution of ions of each feature

Figure in Appendix A.

Table 2 sample ions in the solution relative abundance tolerance range

The relative abundance Tolerance > 50 ± 20 20 ~ 50 ± 25 10 ~ 20 ± 30 <=10 ± 50

7.5 Blank test

But without addition of the sample, the same steps employed in parallel operation.

8 and the result of the calculation expression the specimens cyromazine and melamine residues (µg/kg) according to formula (1).

AcSV1V3

ASV2m

Where.

--- for X-try respective feed cyromazine residues or melamine, in micrograms per kilogram (µg/kg);

A --- respective sample solution cyromazine or melamine peak area;

--- respective concentrations cS cyromazine or melamine, units of nanograms per milliliter (ng/mL) matrix-matched standard solution;

Vl --- extract of a total volume of liquid in milliliters (mL);

--- V2 through the solid phase extraction column with the extract volume in milliliters (mL);

V3 --- volume of the residue was dissolved in milliliters (mL);

--- the AS matrix-matched standard solution corresponding cyromazine or melamine peak area;

m --- try supply feed mass in grams (g).

Note. The blank value should be subtracted from the results, expressed as the arithmetic mean of the measurement result measured parallel to three significant figures.

9.1 Sensitivity

The detection limit of the method was 1µg/kg, limit of quantification was 2.5µg/kg.

9.2 Accuracy

This method of adding 2.5µg/kg ~ 100µg/kg on recovery levels of 70% to 120%.

9.3 Precision

<=20% relative standard deviation in this method and inter - batch relative standard deviation of <=20%.

Appendix A

Chromatogram

Figure A.1 cyromazine and melamine standard solution wherein ion mass chromatogram (10ng/mL)

Figure A.2 chicken kidney tissue sample blank wherein the ion mass chromatogram

Description.

1 --- cyromazine wherein ion mass chromatogram (166.9 > 84.6);

2 --- cyromazine wherein ion mass chromatogram (166.9 > 124.8);

3 --- Melamine wherein ion mass chromatogram (126.9 > 67.7);

4 --- Melamine wherein ion mass chromatogram (126.9 > 84.7).

Figure A.3 blank Add chicken kidney tissue sample features of cyromazine and melamine-ion mass chromatogram (10ng/g)